The telomerase enzyme is a potential therapeutic target in many human cancers. A series of potent inhibitors has been designed by computer modeling, which exploit the unique structural features of quadruplex DNA. These 3,6,9-trisubstituted acridine inhibitors are predicted to interact selectively with the human DNA quadruplex structure, as a means of specifically inhibiting the action of human telomerase in extending the length of single-stranded telomeric DNA. The anilino substituent at the 9-position of the acridine chromophore is predicted to lie in a third groove of the quadruplex. Calculated relative binding energies predict enhanced selectivity compared with earlier 3,6-disubstituted compounds, as a result of this substituent. The ranking order of energies is in accord with equilibrium binding constants for quadruplex measured by surface plasmon resonance techniques, which also show reduced duplex binding compared with the disubstituted compounds. The 3,6,9-trisubstututed acridines have potent in vitro inhibitory activity against human telomerase, with EC50 values of up to 60 nM. T he telomeric ends of chromosomes consist of tandem repeats of simple guanine-rich DNA protein-associated motifs whose function is to protect the ends from unwanted DNA damage-repair, recombination, and end-fusions. In eukaryotics the repeat is TTAGGG, with telomere length varying between ca. 5 and 15 kb (1, 2). Cancer cells typically have short telomeres, whereas stem cell telomere length tends to be at the high end of this range. The terminal 150-200 bases at the 3Ј end of human telomeres form a single-stranded overhang, whose exact structure is not fully established, although loop-type arrangements have been suggested from electron microscope studies (3). Telomeres shorten in somatic cells on each round of replication, by 50-200 bases, as a consequence of the inability of DNA polymerase to fully replicate the ends (4). Once telomeres reach a critically short length, cells enter a senescent state and do not replicate further (5). By contrast, the short telomeres in tumor cells are stable in length, maintained by the action of a specialized DNA polymerase, the telomerase enzyme complex, which catalyses the synthesis of further telomere repeats (6). Telomerase is activated in 80-90% of human tumors and is undetectable in most normal somatic cells (7). This activation has been shown to be a key step in the immortalization process in human cells, leading to tumorigenesis (8). A small proportion of tumor cells have an alternative telomere maintenance pathway (ALT) which appears to be independent of telomerase and involves recombination events. Inhibition of telomerase by a dominant negative mutant (9, 10), or by synthetic oligonucleotides targeted to the RNA template (11), leads to telomere shortening, growth arrest and apoptosis for tumor cells in culture. Telomerase is thus a highly attractive target for selective anti-cancer therapy (12).We have focused on the rational discovery of small-molecule telomerase inhibitors with pharma...

The interaction of DAPI and propidium with RNA (polyA.polyU) and corresponding DNA (polydA.polydT) sequences has been compared by spectroscopic, kinetic, viscometric, Tm, and molecular modeling methods. Spectral changes of propidium are similar on binding to the AT and AU sequences but are significantly different for binding of DAPI. Spectral changes for DAPI with the DNA sequence are consistent with the expected groove-binding mode. All spectral changes for complexes of propidium with RNA and DNA and for DAPI with RNA, however, are consistent with an intercalation binding mode. When complexed with RNA, for example, DAPI aromatic protons signals shift significantly upfield, and the DAPI UV-visible spectrum shows significantly larger changes than when complexed with DNA. Slopes of log kd (dissociation rate constants) versus-log [Na+] plots are similar for complexes of propidium with RNA and DNA and for the DAPI-RNA complex and are in the range expected for an intercalation complex. The slope for the DAPI-DNA complex, however, is much larger and is in the range expected for a groove-binding complex. Association kinetics results also support an intercalation binding mode for the DAPI-RNA complex. The viscosity of polyA.polyU solutions increases significantly on addition of both propidium and DAPI, again in agreement with an intercalation binding mode for both molecules with RNA. Molecular modeling studies completely support the experimental findings and indicate that DAPI forms a very favorable intercalation complex with RNA. DAPI also forms a very stable complex in the minor groove of AT sequences of DNA, but the stabilizing interactions are considerably reduced in the wide, shallow minor groove of RNA. Modeling studies,thus,indicate that DAPI interaction energetics are more favorable for minor-groove binding in AT sequences but are more favorable for interaction in RNA.

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Dicationic 2,4-bis(4-amidinophenyl)furans 5-10 and 2, 4-bis(4-amidinophenyl)-3,5-dimethylfurans 14 and 15 have been synthesized. Thermal melting studies revealed high binding affinity of the compounds to poly(dA-dT) and to the duplex oligomer d(CGCGAATTCGCG)2. All of the new compounds were effective against Pneumocystis carinii pneumonia in the immunosuppressed rat model with up to 200-fold increase in activity compared to the control compound pentamidine. No toxicity was noted for 5, 7-10 at the dose of 10 micromol/kg/d; however, the isopropyl analogue 7 showed toxicity comparable to pentamidine at the dosage of 20 micromol/kg/d. Dimethylation of the parent compound on the furan ring resulted in reduced activity and increased toxicity.